Detailed physical maps have been constructed of hybrid lambda bacteriophage carrying EcoR1 fragments cloned from DNA of defective interfering particles (dDNA) of Herpes simplex virus type 1 (HSV1) (strain Patton). Among our 34 cloned isolates, two size classes were identified. The major class (8-9.5 kilo base pairs (kbp)) made up about 80% of the fragments of the dDNA population, while the minor class (6.5-8 kbp) made up the remainder. Members of the two general size classes could be separated into three structural groups. All groups contained restriction sites characteristic of the S region terminal repeat (TRs). Major class Group I contained dDNA EcoR1 fragments with restriction sites characteristic of the right end of the unique S region (Us) in the prototype orientation. This group is typical of HSV dDNA characterized previously. The minor class representatives of Group I contained an apparent 2kb site-specific deletion in the TRs region. Group II contained dDNA EcoR1 fragments with restriction sites characteristic of the left end of the Us.